Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.     

Recognition of pre-processed endogenous antigen by class I but not class II MHC-restricted T cells

Class I and class II MHC-restricted T lymphocytes recognize non-native forms of antigen. The presentation of antigen to these two classes of T lymphocytes can occur through distinct pathways. Several mechanisms, including differences in antigen processing in different intracellular compartments, have been proposed to account for these pathway differences. Here we describe a T-cell epitope located on the influenza virus haemaglutinin, which is recognized by both class I and class II MHC-restricted cytolytic T lymphocytes (CTL). When expressed de novo in target cells, from a synthetic minigene encoding only the epitope, this pre-processed antigenic site is recognized by class I but not class II MHC-restricted T lymphocytes, even though target cells treated with the exogenously introduced peptide can be recognized by both classes of T cells. Because endogenous expression of the pre-processed antigenic fragment results in differential presentation to class I and class II MHC-restricted CTL, differences between the two different pathways of presentation could lie not at the level of processing but at the level of targeting and/or interaction of processed antigen with MHC.     

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.     

HLA-A2 peptides can regulate cytolysis by human allogeneic T lymphocytes

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.     

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.     

Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with beta 2-microglobulin, in vitro

Cytotoxic T lymphocytes kill virally infected cells when they detect antigenic fragments presented by class I major histocompatibility complex (MHC) antigens (HLA in humans). The crystal structures of HLA-A2 and HLA-Aw68 reveal that peptide-antigen forms an integral part of the HLA structure, being retained in a prominent groove even after purification and crystallization. Here we report that the heavy chain and beta 2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, beta 2-microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. Reconstitution is not stimulated by either of two peptides not restricted to HLA-A2. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, Np (85-94), crystallizes under conditions previously used to crystallize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence.     

T细胞功能学与结构免疫学结合方法鉴定HLA-A2限制性的细胞毒性T淋巴细胞表位富集区

 T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

Interleukin-2 production used to detect antigenic peptide recognition by T-helper lymphocytes from asymptomatic HIV-seropositive individuals

T lymphocytes from mice and healthy humans immunized against the human immunodeficiency virus (HIV) envelope have recently been shown to recognize two antigenic regions of the gp160 HIV-envelope protein which have been located on the basis of amphipathicity. In HIV-infected humans, T-cell proliferative responses are lost soon after infection. Here we demonstrate that interleukin-2 production is often retained even when proliferative activity is absent, and that it can be used to monitor T-helper cell responses by HIV-seropositive donors. We use this approach to investigate the T-helper cell response of 42 asymptomatic HIV-seropositive patients to four synthetic gp160 peptides and to influenza A virus, an antigen requiring intact CD4 T-helper cell function. As many as 67% of the HIV-seropositive donors who retain responsiveness to influenza A virus respond to a single peptide, and 85-90% responded to at least one of the peptides.     

Evolution and transmission of stable CTL escape mutations in HIV infection.

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.     

Screening of HLA-A2 restricted CTL epitopes using molecular simulation

Immunity is one of the functions a body has to iden-tify and eliminate foreign antigens. As one of the surfacemolecules of antigen presenting cells, major histocom-patibility complex (MHC) will exert essential effects dur-ing the antigen presenting procession, and the presenting of MHC-Ⅰ peptide complex is regarded as the prelude to activate the cytotoxic T lymphocytes (CTL) which play an important role in the anti-tumor and anti-virus responses. As we know, CTLs can only identify the co…     

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.     

HLA-A2限制性CTL表位肽定量构效关系研究

基于SCORE打分函数，运用定量构效关系的理论和方法研究了HLA—A2限制性CTL表位九肽结构与亲和性间的定量关系，并建立了SCORE得分与亲和性的定量关系模型，并用外部样本（5个HLA—A2限制性CTL表位九肽）作为预测集用于检验模型的预测能力．基于SCORE打分函数建立的定量模型具有较好的相关性（r=0，9165，RMS=0．38）和对外部样本的预测能力（rpred=0．9847，RMS=0．135）．基于SCORE打分函数，运用定量构效关系研究的理论和方法建立了HLA—A2限制性CTL表位亲和性的定量预测方法，为实验鉴定高亲和性HLA—A2限制性CTL表位提供了理论依据．     

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.     

Structure modeling and spatial epitope analysis for HA protein of the novel H1N1 influenza virus

In recent months, a novel influenza virus H1N1 broke out around the world. With bioinformatics technology, the 3D structure of HA protein was obtained, and the epitope residues were predicted with the method developed in our group for this novel flu virus. 58 amino acids were identified as potential epitope residues, the majority of which clustered at the surface of the globular head of HA protein. Although it is located at the similar position, the epitope of HA protein for the novel H1N1 flu virus has obvious differences in the electrostatic potential compared to that of HA proteins from previous flu viruses.     

In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.     

Peptide binding to empty HLA-B27 molecules of viable human cells

Intracellular binding of antigenic peptides by polymorphic class I major histocompatibility complex molecules creates the ligands recognized by receptors of CD8+ T cells. Previously described in vitro assays of peptide binding to class I molecules have been limited by either the low proportion of accessible binding sites or the lack of allelic specificity. Here we describe a system in which the human class I molecule HLA-B27 binds considerable amounts of an influenza peptide with precise allelic discrimination. Binding requires viable cells, is stimulated by gamma-interferon and is inhibited by brefeldin A. Our results are consistent with the presence of fairly stable 'empty' HLA-B27 molecules at the cell surface. By contrast, analysis of the binding of a second influenza peptide indicates that empty HLA-Aw68 molecules are relatively short-lived. We speculate that HLA-B27 might bind extracellular peptides in vivo and that this property could underlie its association with autoimmune disease.     

Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the alpha 2 domain of HLA-A2

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.